Changes in the repolarization properties of cardiac muscle frequently underlie the increased susceptibility to ventricular arrhythmias in a variety of heart diseases, but the control of cardiac repolarization is incompletely understood. Abnormalities of repolarization resulting in action potential duration prolongation or shortening can be pro-arrhythmic. Multiple different ion channels in cardiomyocytes impact action potential repolarization, and the diversity of ion channels and regulatory pathways involved in repolarization is highlighted by the association of at least 13 distinct genes with the inherited long QT syndrome which is due to delayed repolarization. For example, mutations in the caveolin-3 gene (CAV3) have been implicated as one cause of the inherited Long QT syndrome (LQTS). Cav-3 is an essential scaffolding protein required for the formation of specialized membrane microdomains in cells referred to as caveolae which are home to multiple signaling molecules and ion channel proteins. Preliminary data presented in this application show that genetic ablation of caveolin-3 in the mouse heart dramatically prolongs cardiac repolarization. The proposed research will test the hypothesis that cardiac caveolae provide integrated regulation of cardiac repolarization by controlling the density and functional properties of specifc ion channels. Using both genetically engineered mouse models with cardiac-specific regulation of Cav-3 expression as well as human iPS cell-cardiomyocyte models, the proposal will examine the role of caveolae in the regulation of cardiac repolarization in three specific aims: 1) Determine the impact of changes in the abundance of Cav-3 in cardiomyocytes on the density of caveolae and on cardiac repolarization; 2) Evaluate the impact of changes in Cav-3 abundance and LQTS-associated Cav-3 mutations on the density and biophysical properties of voltage-gated potassium currents; and 3) Determine how wild type Cav-3 and LQTS-associated mutations of Cav-3 regulate the density and properties of ICa,L. These studies will provide mechanistic new insights into the control of cardiac repolarization and abnormalities in disease which can result in arrhythmias.